Clathrin-mediated post-fusion membrane retrieval influences the exocytic mode of endothelial Weibel-Palade bodies.

Nicola L Stevenson,Thomas D Nightingale,Daniel F Cutler,Jessica J Mccormack,Christopher Robinson,Ian J White

doi:10.1242/jcs.200840

Abstract

ABSTRACTWeibel-Palade bodies (WPBs), the storage organelles of endothelial cells, are essential to normal haemostatic and inflammatory responses. Their major constituent protein is von Willebrand factor (VWF) which, following stimulation with secretagogues, is released into the blood vessel lumen as large platelet-catching strings. This exocytosis changes the protein composition of the cell surface and also results in a net increase in the amount of plasma membrane. Compensatory endocytosis is thought to limit changes in cell size and retrieve fusion machinery and other misplaced integral membrane proteins following exocytosis; however, little is known about the extent, timing, mechanism and precise function of compensatory endocytosis in endothelial cells. Using biochemical assays, live-cell imaging and correlative spinning-disk microscopy and transmission electron microscopy assays we provide the first in-depth high-resolution characterisation of this process. We provide a model of compensatory endocytosis based on rapid clathrin- and dynamin-mediated retrieval. Inhibition of this process results in a change of exocytic mode: WPBs then fuse with previously fused WPBs rather than the plasma membrane, leading, in turn, to the formation of structurally impaired tangled VWF strings.This article has an associated First Person interview with the first authors of the paper.

Highlights

Many cell types utilise regulated secretion as a means to release premade bioactive material from membranous carriers at the cell surface (Burgoyne and Morgan, 2003)
If the purpose of Weibel-Palade bodies (WPBs) compensatory endocytosis is solely to retrieve membrane this could be carried out anywhere on the plasma membrane and begs the question as to why clathrincoated pits are found on fused structures containing von Willebrand factor (VWF). We investigated this process in human umbilical vein endothelial cells (HUVECs) using biochemical assays, transmission electron microscopy (TEM) and correlative live-cell imaging and TEM to define the extent, mode, mechanism and function of compensatory endocytosis
There are a large number of secretagogues that stimulate WPB exocytosis – some of which trigger a Ca2+-dependent release and some of which act via cAMP (Rondaij et al, 2006) – and phorbol 12-myristate 13-acetate (PMA) uses both; we wanted to monitor the effect on endocytosis irrespective of the route of stimulation

Summary

Introduction

Many cell types utilise regulated secretion as a means to release premade bioactive material from membranous carriers at the cell surface (Burgoyne and Morgan, 2003) These include soluble factors for release into the extracellular milieu, as well as integral membrane proteins that are displayed for interaction with their cognate ligands. Compensatory endocytosis provides a means to limit this membrane expansion to maintain cell size and membrane tension, as well as to return key integral membrane proteins back into the cell (Gordon and Cousin, 2016; Reider and Wendland, 2011) This retrieval process is best understood in neurons and neuroendocrine cells where a number of discrete mechanisms occur. This process requires dynamin (Henkel and Almers, 1996; Artalejo et al, 1995; Holroyd et al, 2002) and calcium (Henkel and Almers, 1996), and in some cases allows differential release of cargo based on the size of the fusion pore (Barg and Machado, 2008)

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